Pharmaceutical compositions of goserelin sustained release microspheres

ABSTRACT

A composition of goserelin sustained release microspheres is provided. The microspheres comprise goserelin, at least one poly(lactide-co-glycolide) and poloxamer or PEG. The sustained release microspheres have comparatively high bioavailability, which promotes the drug taking its full effect and have entrapment efficiency over 90%.

CROSS-REFERENCE TO RELATED APPLICATIONS

The application is a continuation application of InternationalApplication No. PCT/CN2014/075441, filed Apr. 16, 2014, which claimspriority to Chinese Application No. 201310136505.6, filed Apr. 18, 2013,the contents of each of which are incorporated by reference in theirentireties for all purposes.

FIELD OF THE INVENTION

The present disclosure relates to pharmaceutical preparations and, moreparticularly, to compositions of long-acting sustained release goserelinmicrospheres, methods for preparing the same and use of the same.

BACKGROUND OF THE INVENTION

Gonadotropin-releasing hormone (GnRH), also known as luteinizinghormone-releasing hormone (LHRH), is a hormone closely related toreproductive functions. When an exogenous LHRH or an analogue thereof isadministrated with a physiological pulse frequency (once per 90 min) fora short period of time and at a small dose, it produces some promotingeffects to the pituitary-gonadal system, and hence is used clinicallyfor treating symptoms such as sexual dysfunction, anovulation, delayedpuberty, etc. When it is administrated with a non-physiological pulsefrequency for a long period of time and at a large dose, it can inhibitthe hypophysis from secreting luteinizing hormone (LH) and folliclestimulating hormone (FSH), resulting in a decrease in the hormonesecretion capacity of gonad and the atrophy of sexual organs. It is thusused clinically for treating some hormone-dependent diseases such asprostate cancer, hysteromyoma, breast carcinoma, adenomyosis, precociouspuberty, etc.

Analogues of LHRH regulate the secretion of LH and FSH through feedbackinhibition by competitively binding to most of the hypophyseal LHRHreceptors, whereby inhibit the produce of ovarian estrogen, and achievetherapeutic effect of medical oophorectomy. Studies have shown thatlives of prostate cancer patients can be prolonged by administeringhormone analogues of LHRH (such as goserelin) after radiotherapy.Available information has shown that hormone analogues of LHRH are atleast able to achieve the same therapeutic effect as surgical castrationor CMF chemotherapy, and are used as postoperative adjuvant treatmentfor premenopausal patients with breast cancer; and achieve the sametherapeutic effect as CMF chemotherapy in estrogen receptor-positivepatients with axillary lymph node metastasis, with fewer side effects,and more acceptable to the patients. In recent years, goserelin has beenused for control of clinical signs and symptoms of endometriosis andadenomyosis to prevent recurrence of endometriosis after surgery, allobtained good results. In addition, goserelin has also been used forendometrium thinning and treatment of uterine fibroids and othersymptoms. Several clinical products of LHRH analogues have beencommercially available. For example, the goserelin preparation, with thetrade name of “Zoladex”, has been approved in France in 1987 andapproved by FDA On Dec. 29, 1989. Its dosage form is an implant, with amonthly injection dose of 3.6 mg/vial. Adults are subcutaneouslyinjected 3.6 mg once every 28-days at anterior abdominal. However, “forthe subcutaneous injection of Zoladex at anterior abdominal, the stypeis preloaded in a disposable syringe, and the syringe needle isequivalent in size to the size 16 puncture needle which is 30 mm long,therefore as compared with subcutaneous injection of common drugs, thedegree of pain caused by injection of Zoladex is higher and bleedingcaused by injection of Zoladex occurs more often.”—Journal of ModernClinical Medical, July, 2008, Volume 6 (7).

According to the characteristics of administration of goserelin to itsclinical indications, patients often require long-term administration.Thus, in order to improve patient compliance, goserelin has beendeveloped into long-acting sustained release preparations. Compared withthe implant preparations, injecting microsphere preparations to patientssignificantly reduces the patients' pain and bleeding. Severalcommercially available LHRH analogues microspheres, such as leuprolidemicrospheres, adopt this drug release pattern. However, studies haveshown that when microspheres are prepared from goserelin to which nopre-treatment is subjected, the drug entrapment efficiency is low andthe loss in the manufacturing process is high, resulting in increasingin production cost. Pharmacokinetic studies of the prepared microspheresin animals have shown that the bioavailability of goserelin is low,which makes the drug incapable of acting to its full effect.

SUMMARY OF THE INVENTION

Through in-depth research, we found that by pretreating goserelin withadded poloxamer or polyethylene glycol (PEG) therein and then preparingmicrospheres from the pretreated goserelin increases the drug entrapmentefficiency and improves the bioavailability of the drug, which promotesthe drug taking its full effect.

The present disclosure provides a pharmaceutical composition ofsustained release goserelin microspheres. The goserelin microspherescomprise (a) goserelin or a salt thereof, (b) copolymers of lactide andglycolide (poly(lactide-co-glycolide; PLGA), and (c) poloxamer orpolyethylene glycol (PEG).

In the pharmaceutical composition of sustained release goserelinmicrospheres of the present disclosure, the content by weight ofgoserelin or the salt thereof may be 1-10%, preferably 1-8%, morepreferably 1-5%. The content by weight of the copolymers of lactide andglycolide may be 80-98%, preferably 86-98%, more preferably 91-98%. Thecontent by weight of the poloxamer or polyethylene glycol may be 1-10%,preferably 1-6%, more preferably 1-4%.

The pharmaceutical composition may further comprise less than 0.1% byweight of (d) acetic acid, preferably less than 0.01%, more preferably0.008%.

In one pharmaceutical composition, the weight content of the goserelinis 1-10%; the weight content of the poly(lactide-co-glycolide) is80-98%; and the weight content of the poloxamer or PEG is 1-10%.

In another pharmaceutical composition, the weight content of thegoserelin is 1-8%; the weight content of the poly(lactide-co-glycolide)is 86-98%; and the weight content of the poloxamer or PEG is 1-6%.

In yet another pharmaceutical composition, the weight content of thegoserelin is 1-5%; the weight content of the poly(lactide-co-glycolide)is 91-98%; and the weight content of poloxamer or PEG is 1-4%.

The copolymer of lactide and glycolide is also referred to aspoly(lactide-co-glycolide), abbreviated as PLGA. The molar ratio oflactide to glycolide in said PLGA may be 90:10 to 10:90, preferably75:25 to 25:75, more preferably 60:40 to 40:60, most preferably 50:50.

The intrinsic viscosity of poly(lactide-co-glycolide) (PLGA) may be0.10-0.40 dug, preferably in the range of 0.10-0.35 dL; g, morepreferably in the range of 0.10-0.30 dL/g. A method for measuring theintrinsic viscosity of PLGA may be as follows: prepare an about 0.5%(w/v) solution of PLGA in chloroform, and determine the intrinsicviscosity of PLGA at 30° C. using a Cannon-Fenske glass capillaryviscometer.

The PLGA described in the present disclosure may have a molecular weightof 4,000-45,000 Dalton, preferably 4,000-35,000 Dalton, more preferably4,000-30,000 Dalton. As used herein, the term “molecular weight” refersto “weight average molecular weight.”

For convenience of description, the molar ratio of lactide to glycolide,the intrinsic viscosity and molecular weight of PLGA are shownhereinafter in brackets following each PLGA. For example, “PLGA (50/50,0.14, 7,200)” represents poly(lactide-co-glycolide) with a molar ratioof lactide to glycolide of 50:50, an intrinsic viscosity of 0.14 dL/g,and a molecular weight of 7,200 Dalton.

The poloxamers described in the present disclosure are block copolymersof polyethylene glycol and polypropylene glycol. The copolymers arecomposed of various compounds with different hydrophile-lipophilebalance and are formed by appropriate amount of polyoxypropylene andappropriate amount of polyoxyethylene. The preferred poloxamers isPoloxamer 188 or Poloxamer 407, more preferably Poloxamer 188.

The polyethylene glycol (PEG) described in the present disclosure, alsoknown as polymer of α-hydrogen-ω-hydroxy(oxy-1,2-ethanediyl) orpolyoxyethylene (PEO-LS), is a general term for glycol polymer. Thepolyethylene glycol may be PEG 2000, PEG 4000 or PEG 6000, preferablyPEG 6000.

The drug loading amount is the actual drug loading amount, which may becalculated as follows: drug loading amount=[amount of drug inmicrospheres/(amount of drug in microspheres+amount of polymericexcipients)]×100% (I), wherein the polymeric excipients include polymersdisclosed in the present disclosure, for example the combination of PLGAand poloxamers or the combination of PLGA and polyethylene glycol.

The entrapment efficiency may be calculated as follows: entrapmentefficiency (%)=[measured drug loading amount inmicrospheres/(theoretical drug loading amount in to microspheres)]×100%(II), wherein the “measured drug loading amount in microspheres” informula (II) is the “drug loading amount” calculated according toformula (I); wherein the “theoretical drug loading amount inmicrospheres” is calculated according to formula (I) based on the amountof the drug and the amount of the polymeric excipients measuredseparately before they are combined for the preparation of microspheres.

The salt of goserelin in the sustained release microspheres provided bythe present disclosure may be any water-soluble salt such as an acetatesalt.

For each pharmaceutical composition of the sustained release goserelinmicrospheres of the present disclosure, a preparation method isprovided. The method involves a solid-in-oil-in-water (s/o/w)emulsion-solvent evaporation process. The process comprises pre-treatinggoserelin acetate with poloxamer or PEG, and then adding the pretreatedgoserelin acetate to an oil phase comprising poly(lactide-co-glycolide).The poloxamer or PEG pre-treated goserelin acetate in the oil phase mayhave a particle size d (0.5) of 0.01-2 μm.

Goserelin acetate may be pretreated by mixing the goserelin acetate withpoloxamer or polyethylene glycol to form a solid powder mixture; PLGA isdissolved in an organic solvent to form an oil phase; the solid powdermixture is added into the oil phase, and then subjected to shearingemulsification to obtain a solid-in-oil (s/o) primary emulsion. Then,the primary emulsion is added into an aqueous solution containing anemulsifier, homogeneously emulsified to obtain an S/O/W double emulsion,then the organic solvent is removed from the S/O/W double emulsion, andthe residue is washed and filtered to obtain the microspheres.

The organic solvent may be ethyl acetate, chloroform or dichloromethane,preferably dichloromethane.

The emulsifiers may be hydrophilic emulsifiers, for example, tweens,polyethylene glycol octylphenol ether (Triton), Brij, Poly polyvinylpyrrolidone or polyvinyl alcohol, preferably polyvinyl alcohol (PVA).

The concentration of emulsifier in the aqueous solution may be 0.01%-5%,preferably 0.02-2%, more preferably 0.5%-1.0%.

The present disclosure provides a use of the goserelin microspheres inpreparation of drugs for treating prostate cancer, sexual precocity,adenomyosis, female infertility, and hysteromyoma.

The present disclosure also provides a method for treating prostatecancer, sexual precocity, adenomyosis, female infertility, orhysteromyoma in a subject in need thereof. The treatment methodcomprises administering to the subject an effective amount of thepharmaceutical composition of sustained release goserelin microspheresdescribed in the present disclosure. The subject may be an animal,preferably a mammal, more preferably a human. The pharmaceuticalcomposition of the present invention may be formulated for oral,sublingual, intranasal, intraocular, rectal, transdermal, mucosal,topical or parenteral administration. Parenteral administration mayinclude intradermal, subcutaneous (s.c., s.q., sub-Q, Hypo),intramuscular (i.m.), intravenous (i.v.), intraperitoneal (i.p.),intra-arterial, intramedulary, intracardiac, intra-articular (joint),intrasynovial (joint fluid area), intracranial, intraspinal, andintrathecal (spinal fluids) injection or infusion, preferablyintraperitoneal (i.p.) injection in mouse and intravenous (i.v.) inhuman. Any device suitable for parenteral injection or infusion of drugformulations may be used for such administration. For example, thepharmaceutical composition may be contained in a sterile pre-filledsyringe.

The microspheres provided by the present invention may be made into theform of sterile powder, wherein the sterile powder may comprise thecomposition of goserelin microspheres and mannitol, and may be preparedas follows: rinsing the composition of goserelin microspheres with waterfor injection and transferring into a freeze-drying tray, addingmannitol and a proper amount of water for injection therein, placing thefreeze-drying tray in a freeze drier for freeze-drying; and subjectingthe freeze-dried product to sieving and mixing, aseptic filling andcapping, so as to obtain the sterile powder. Before being administratedto a patient, the sterile powder may be suspended in a pharmaceuticallyacceptable dispersion solvent, wherein the dispersion solvent maycomprise a suspending agent, a pH regulator, an isoosmotic adjustingagent, a surfactant, or a combination thereof, together with water forinjection; the suspending agent may be selected from the groupconsisting of sodium carboxymethyl cellulose, polyvinyl alcohol,polyvinylpyrrolidone, sodium alginate, glycerol, and a combinationthereof; the isoosmotic adjusting agent may be selected from the groupconsisting of sodium chloride, glucose, mannitol, sorbitol and acombination thereof; and the surfactant may be a nonionic surfactantsuch as polysorbate series (e.g., polysorbate 80, polysorbate 60, etc.).The sustained release goserelin microspheres provided by the presentdisclosure may be used for intramuscular or subcutaneous injection.

The sustained release goserelin microspheres described in the presentdisclosure may have high entrapment efficiency over, for example, 80%,90%, 95% or 99%, preferably 90%, when goserelin is pretreated bypoloxamer or PEG, and may have relatively high bioavailability in vivoof, for example, at least 1%, 5%, 10%, 20%, 30%, 40% or 50% in a subjectafter administration.

A pharmaceutical composition of sustained release goserelin microspheresaccording to the present invention may have higher entrapment efficiencythan a control composition by, for example, by at least 1%, 5%, 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. The control compositionis identical to the pharmaceutical composition except having no or lesspoloxamer or polyethylene glycol (PEG).

When administered to a subject in need thereof, a pharmaceuticalcomposition of sustained release goserelin microspheres according to thepresent invention may have higher bioavailability in the subject than acontrol composition by, for example, at least 1%, 5%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, or 95%. The control composition isidentical to the pharmaceutical composition except having no or lesspoloxamer or polyethylene glycol (PEG). For example, when administeredto a subject in need thereof, a pharmaceutical composition of thepresent invention having at least 1% poloxamer or polyethylene glycol(PEG) may have at least 20% higher bioavailability in the subject than acontrol composition lacking the poloxamer or polyethylene glycol (PEG).

The particle size span used in the present disclosure is definedaccording to the Guidelines for Microcapsules, Microspheres and LiposomePreparations, Appendix XIXE of Chinese Pharmacopoeia (2010 Edition) asfollows:

Span=(D90−D10)/D50

wherein, D90, D50 and D10 refer to the particle sizes respectivelycorresponding to 90%, 50% and 10% of the cumulative distribution in thecumulative curve. The smaller the span is, the narrower the distributionand thus the more uniform the particle size.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of rat in vive drug release curves of goserelinmicrospheres which contain different amount of poloxamer/PEG.

FIG. 2 is a graph of rat in vivo drug release curves of goserelinmicrospheres which contain different amount of poloxamer or PEG.

FIG. 3 is a graph of rat in vivo drug release curves of goserelinmicrospheres without control of the acetic acid content before and afterthe 6-month stability test.

FIG. 4 is a graph of rat in vivo drug release profiles of goserelinmicrospheres with control of the acetic acid content before and afterthe 6-month stability test.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure will be further illustrated by the followingexamples and test examples, which will not limit the scope of thepresent invention in any way.

Example 1

Appropriate amount of goserelin acetate and Poloxamer 188 were weighedand ball-mill mixed at frequency of 15 Hz for 5 min so as to obtain amixture of solid powder. 430 mg mixture of goserelin and Poloxamer 188(the measured amount of goserelin was 215 mg) was accurately weighed forlater use. 1.721 g of PLGA (75/25, 0.35, 42,000) was weighed anddissolved in 10 ml of dichloromethane to form an oil phase; and then thepretreated drug mixture of solid powder was added into the oil phase,and subjected to emulsification in a high shear emulsifier (6,500 rpm, 3min) so as to obtain a s/o primary emulsion. The primary emulsion wasadded into 1,000 ml of a 0.5% PVA solution at 6° C. under homogenizationat 1,800 rpm, and then it was homogeneously emulsified for 2 min toobtain an S/O/W double emulsion. The double emulsion was stirred tovolatilize and remove the organic solvent; the residue was washed andfreeze-dried to obtain powdery microspheres. The microspheres had a drugloading amount of 9.01% and an entrapment efficiency of 90.1%.

Example 2

The melting temperature of a melt extruder was set at 80° C. Appropriateamount of goserelin acetate and Poloxamer 407 were sifted and mixed. Themixture was fed into the cavity of the extruder. The stirring speed wasset to n=60 and the stirring mixing time to 3 min. Then the valve handlewas released to extrude the melted material, which was then allowed tobecome cool naturally. The material was ball-mill smashed for 2 min. 316mg of the mixture of goserelin and Poloxamer 188 (the measured amount ofgoserelin was 158 mg) was accurately weighed for later use, 1.672 g ofPLGA (25/75, 0.24, 25,000) was weighed and dissolved in 10 ml ofdichloromethane to form an oil phase; and then the pretreated drugmixture of solid powder was added into the oil phase, and subjected toemulsification in a high shear emulsifier (6,500 rpm, 3 min) so as toobtain a s/o primary emulsion. The primary emulsion was added into 1,000ml of a 0.5% PVA solution at 6° C. under homogenization at 1,800 rpm,and then it was homogeneously emulsified for 2 min to obtain an S/O/Wdouble emulsion. The double emulsion was stirred to volatilize andremove the organic solvent; the residue was washed and freeze-dried toobtain powdery microspheres. The microspheres had a drug loading amountof 7.21% and an entrapment efficiency of 90.7%.

Example 3

Appropriate amount of goserelin acetate and Poloxamer 188 were weighedand dissolved in water to form a clear solution, and then the solutionwas sprayed dried so as to obtain a mixture of solid powder. 47 mgspray-dried mixture of goserelin and Poloxamer 188 (the measured amountof goscrelin was 23 mg) was accurately weighed and put into a vial.1.951 g of PLGA (65/35, 0.29, 32,000) was weighed and dissolved in 10 mlof dichloromethane to form an oil phase; and then the pretreated drugmixture of solid powder was added into the oil phase, and subjected toemulsification in a high shear emulsifier (6,500 rpm, 3 min) so as toobtain a s/o primary emulsion. The primary emulsion was added into 1,000ml of a 0.5% PVA solution at 6° C. under homogenization at 1,800 rpm,and then it was homogeneously emulsified for 2 min to obtain an S/O/Wdouble emulsion. The double emulsion was stirred to volatilize andremove the organic solvent; the residue was washed and freeze-dried toobtain powdery microspheres. The microspheres had a drug loading amountof 1.02% and an entrapment efficiency of 90.1%.

Example 4

92 mg of goserelin acetate (containing 80 mg of goserelin as measured)and 21 mg of Poloxamer 188 were weighed and dissolved in 10 ml of waterto form a clear solution, and then the solution was freeze-dried so asto obtain a mixture of solid powder. 1.908 g of PLGA (50/50, 0.14,7,200) was weighed and dissolved in 10 ml of dichloromethane to form anoil phase; and then the pretreated drug mixture of solid powder wasadded into the oil phase, and subjected to emulsification in a highshear emulsifier (6,500 rpm, 3 min) so as to obtain a s/o primaryemulsion. The primary emulsion was added into 1,000 ml of a 0.5% PVAsolution at 6° C. under homogenization at 1,800 rpm, and then it washomogeneously emulsified for 2 min to obtain an S/O/W double emulsion.The double emulsion was stirred to volatilize and remove the organicsolvent; the residue was washed and freeze-dried to obtain powderymicrospheres. The microspheres had a drug loading amount of 3.62% and anentrapment efficiency of 91.4%.

Example 5

83 mg of goserelin acetate (containing 72 mg of goserelin as measured)and 41 mg of Poloxamer 188 were weighed and dissolved in 10 ml of waterto form a clear solution, and then the solution was freeze-dried so asto obtain a mixture of solid powder. 1.876 g PLGA (50/50, 0.14, 7,200)was weighed and dissolved in 10 ml of dichloromethane to form an oilphase; and then the pretreated drug mixture of solid powder was addedinto the oil phase, and subjected to emulsification in a high shearingemulsifier (6500 rpm, 3 min) so as to obtain a s/o primary emulsion. Theprimary emulsion was added into 1,000 ml of a 0.5% PVA solution at 6° C.under homogenization at 1,800 rpm, and then it was homogeneouslyemulsified for 2 min to obtain an S/O/W double emulsion. The doubleemulsion was stirred to volatilize and remove the organic solvent; theresidue was washed and freeze-dried to obtain powdery microspheres. Themicrospheres had a drug loading amount of 3.56% and an entrapmentefficiency of 98.6%.

Example 6

91 mg of goserelin acetate (containing 79 mg of goserelin as measured)and 101 mg of Poloxamer 188 were weighed and dissolved in 20 ml of waterto form a clear solution, and then the solution was freeze-dried so asto obtain a mixture of solid powder. 1.811 g PLGA (50/50, 0.14, 7,200)was weighed and dissolved in 10 ml of dichloromethane to form an oilphase; and then the pretreated drug mixture of solid powder was addedinto the oil phase, and subjected to emulsification in a high shearingemulsifier (6,500 rpm, 3 min) so as to obtain a s/o primary emulsion.The primary emulsion was added into 1,000 ml of a 0.5% PVA solution at6° C. under homogenization at 1,800 rpm, and then it was homogeneouslyemulsified for 2 min to obtain an S/O/W double emulsion. The doubleemulsion was stirred to volatilize and remove the organic solvent; theresidue was washed and freeze-dried to obtain powdery microspheres. Themicrospheres had a drug loading amount of 3.61% and an entrapmentefficiency of 91.3%.

Example 7

92 mg of goserelin acetate (containing 80 mg of goserelin as measured)and 201 mg of Poloxamer 188 were weighed and dissolved in 20 ml of waterto form a clear solution, and then the solution was freeze-dried so asto obtain a mixture of solid powder. 1.723 g PLGA (50/50, 0.14, 7,200)was weighed and dissolved in 10 ml of dichloromethane to form an oilphase; and then the pretreated drug mixture of solid powder was addedinto the oil phase, and subjected emulsification in a high shearingemulsifier (6,500 rpm, 3 min) so as to obtain a s/o primary emulsion.The primary emulsion was added into 1000 ml of a 0.50%/PVA solution at6° C. under homogenization at 1800 rpm, and then it was homogeneouslyemulsified for 2 min to obtain an S/O/W double emulsion. The doubleemulsion was stirred to volatilize and remove the organic solvent; theresidue was washed and freeze-dried to obtain powdery microspheres. Themicrospheres had a drug loading amount of 3.58% and an entrapmentefficiency of 90.2%.

Example 8

62 mg of goserelin acetate (containing 54 mg of goserelin as measured)and 41 mg of Poloxamer 188 were weighed and dissolved in 10 ml of waterto form a clear solution, and then the solution was freeze-dried so asto obtain a mixture of solid powder. PLGA (50/50, 0.14, 7,200) and PLGA(0/50, 0.20, 18,000) with weight ratio of 1:1 were weighed and a totalof 1.964 g of PLGAs were dissolved in 10 ml of dichloromethane to forman oil phase; and then the pretreated drug mixture of solid powder wasadded into the oil phase, and subjected to emulsification in a highshearing emulsifier (6,500 rpm, 3 min) so as to obtain a s/o primaryemulsion. The primary emulsion was added into 1,000 ml of a 0.5% PVAsolution at 6° C. under homogenization at 1,800 rpm, and then it washomogeneously emulsified for 2 min to obtain an S/O/W double emulsion.The double emulsion was stirred to volatilize and remove the organicsolvent; the residue was washed and freeze-dried to obtain powderymicrospheres. The microspheres had a drug loading amount of 2.54% and anentrapment efficiency of 97.3%.

Example 9

26 mg of goserelin acetate (containing 23 mg of goserelin as measured)and 58 mg of Poloxamer 188 were weighed and dissolved in 10 ml of waterto form a clear solution, and then the solution was freeze-dried so asto obtain a mixture of solid powder. PLGA (50/50, 0.14, 7,200) and PLGA(0/50, 0.20, 18,000) with weight ratio of 1:3 were weighed and a totalof 1.801 g PLGAs were dissolved in 10 ml of dichloromethane to form anoil phase; and then the pretreated drug mixture of solid powder wasadded into the oil phase, and subjected to emulsification in a highshearing emulsifier (6,500 rpm, 3 min) so as to obtain a s/o primaryemulsion. The primary emulsion was added into 1,000 ml of a 0.5% PVAsolution at 6° C. under homogenization at 1,800 rpm, and then it washomogeneously emulsified for 2 min to obtain an S/O/W double emulsion.The double emulsion was stirred to volatilize and remove the organicsolvent; the residue was washed and freeze-dried to obtain powderymicrospheres. The microspheres had a drug loading amount of 1.15% and anentrapment efficiency of 95.8%.

Example 10

129 mg of goserelin acetate (containing 112 mg of goserelin as measured)and 82 mg of Poloxamer 188 were weighed and dissolved in 10 ml of waterto form a clear solution, and then the solution was freeze-dried so asto obtain a mixture of solid powder. PLGA (50/50, 0.14, 7,200) and PLGA(50, 0.20, 18,000) with weight ratio of 3:1 were weighed and a total of1.861 g PLGAs were dissolved in 10 ml of dichloromethane to form an oilphase; and then the pretreated drug mixture of solid powder was addedinto the oil phase, and subjected to emulsification in a high shearingemulsifier (6,500 rpm, 3 min) so as to obtain a s/o primary emulsion.The primary emulsion was added into 1,000 ml of a 0.5% PVA solution at6° C., under homogenization at 1,800 rpm, and then it was homogeneouslyemulsified for 2 min to obtain an S/O/W double emulsion. The doubleemulsion was stirred to volatilize and remove the organic solvent; theresidue was washed and freeze-dried to obtain powdery microspheres. Themicrospheres had a drug loading amount of 5.31% and an entrapmentefficiency of 98.0%.

Example 11

91 mg of goserelin acetate (containing 79 mg of goserelin as measured)and 6 mg of Poloxamer 188 were weighed and dissolved in 10 ml of waterto form a clear solution, and then the solution was freeze-dried so asto obtain a mixture of solid powder. 1.924 g PLGA (50/50, 0.14, 7,200)was weighed and dissolved in 10 ml of dichloromethane to form an oilphase; and then the pretreated drug mixture of solid powder was addedinto the oil phase, and subjected to emulsification in a high shearingemulsifier (6,500 rpm, 3 min) so as to obtain a s/o primary emulsion.The primary emulsion was added into 1,000 ml of a 0.5% PVA solution at6° C. under homogenization at 1,800 rpm, and then it was homogeneouslyemulsified for 2 min to obtain an S/O/W double emulsion. The doubleemulsion was stirred to volatilize and remove the organic solvent; theresidue was washed and freeze-dried to obtain powdery microspheres. Themicrospheres had a drug loading amount of 2.98% and an entrapmentefficiency of 76.1%.

Example 12

93 mg of goserelin acetate (containing 81 mg of goserelin as measured)and 41 mg of PEG6000 were weighed and dissolved in 10 ml of water toform a clear solution, and then the solution was freeze-dried so as toobtain a mixture of solid powder. 1.931 g PLGA (50/50, 0.14, 7,200) wasweighed and dissolved in 10 ml of dichloromethane to form an oil phase;and then the pretreated drug mixture of solid powder was added into theoil phase, and subjected to emulsification in a high shearing emulsifier(6,500 rpm, 3 min) so as to obtain a s/o primary emulsion. The primaryemulsion was added into 1,000 ml of a 0.5% PVA solution at 6° C. underhomogenization at 1,800 rpm, and then it was homogeneously emulsifiedfor 2 min to obtain an S/O/W double emulsion. The double emulsion wasstirred to volatilize and remove the organic solvent; the residue waswashed and freeze-dried to obtain powdery microspheres. The microsphereshad a drug loading amount of 3.64% and an entrapment efficiency of92.9%.

Example 13

90 mg of goserelin acetate (containing 78 mg of goserelin as measured)and 42 mg of PEG4000 were weighed and dissolved in 10 ml of water toform a clear solution, and then the solution was freeze-dried so as toobtain a mixture of solid powder. 1.925 g PLGA (85/15, 0.36, 44,000) wasweighed and dissolved in 10 ml of dichloromethane to form an oil phase.The pretreated drug mixture of solid powder was added into the oilphase, and then subjected to emulsification in a high shearing (6,500rpm, 3 min) so as to obtain a s/o primary emulsion. The primary emulsionwas added into 1,000 ml of a 0.5% PVA solution at 6° C. underhomogenization at 1,800 rpm, and then it was homogeneously emulsifiedfor 2 min to obtain an S/O/W double emulsion. The double emulsion wasstirred to volatilize and remove the organic solvent; the residue waswashed and freeze-dried to obtain powdery microspheres. The microsphereshad a drug loading amount of 3.43% and an entrapment efficiency of90.1%.

Example 14

94 mg of goserelin acetate (containing 82 mg of goserelin as measured)and 42 mg of PEG2000 were weighed and dissolved in 20 ml of water toform a clear solution, and then the solution was freeze-dried so as toobtain a mixture of solid powder. 1.923 g PLGA (10/90, 0.27, 29,000) wasweighed and dissolved in 10 ml of dichloromethane to form an oil phase;and then the pretreated drug mixture of solid powder was added into theoil phase, and subjected to emulsification in a high shearing emulsifier(6,500 rpm, 3 min) so as to obtain a s/o primary emulsion. The primaryemulsion was added into 1,000 ml of a 0.5% PVA solution at 6° C. underhomogenization at 1,800 rpm, and then it was homogeneously emulsifiedfor 2 min to obtain an S/O/W double emulsion. The double emulsion wasstirred to volatilize and remove the organic solvent; the residue waswashed and freeze-dried to obtain powdery microspheres. The microsphereshad a drug loading amount of 3.59% and an entrapment efficiency of90.4%.

Comparative Example 1

93 mg of goserelin acetate (containing 81 mg of goserelin as measured)was weighed and dissolved in 4 ml of water to form a clear solution, andthen the solution was freeze-dried so as to obtain a mixture of solidpowder. 1.908 g PLGA (50/50, 0.14, 7,200) was weighed and dissolved in10 ml of dichloromethane to form an oil phase; and then the pretreateddrug mixture of solid powder was added into the oil phase, and subjectedto emulsification in a high shearing emulsifier (6,500 rpm, 3 min) so asto obtain a s/o primary emulsion. The primary emulsion was added into1,000 ml of a 0.5% PVA solution at 6° C., under homogenization at 1,800rpm, and then it was homogeneously emulsified for 2 min to obtain anS/O/W double emulsion. The double emulsion was stirred to volatilize andremove the organic solvent; the residue was washed and freeze-dried toobtain powdery microspheres. The microspheres had a drug loading amountof 2.12% and an entrapment efficiency of 52.4%.

Test Example 1 Entrapment Efficiency Tests of Examples and ComparativeExamples

Test method: 25 mg of goserelin reference was weighed and dissolved indistilled water to form a goserelin solution at a concentration of 0.02mg/ml to be used as reference solution. In a 10 ml of volumetric flask,20 mg of goserelin microspheres was dissolved in appropriate amount ofpure acetic acid and diluted with distilled water to reach the volume tomake a test solution. Then the sample was subjected to high speedcentrifugation, and the supernatant was directly injected into highperformance liquid chromatography (HPLC).

The chromatographic column: C₁₈ column (25 cm×4.6 mm, 5 μm);

column temperature: 40° C.±0.5° C.

Mobile phase: 0.5% of phosphoric acid acetonitrile solution-0.59% ofphosphoric acid solution (25:75) (V:V)

Flow rate: 1.0 ml/min;

Detection wavelength: 220 nm;

Sampling volume: 10 μl

10 μl of the test solution and 10 μl of the reference solution wereinjected into the liquid chromatograph. Then the chromatographic peakretention time and chromatographic peak areas were measured and recordedto calculate the amount of goserelin in microspheres by externalstandard method, and the entrapment efficiency can be calculated throughthe aforesaid formula. The entrapment efficiency of the test examplesand the reference sample are shown in Table 1.

TABLE 1 Results of entrapment efficiency in test examples and Controlgroups Entrapment Amount of Poloxamer Example No. Efficiency or PEGExample 1 90.1% 10.00% Example 2 90.7% 7.95% Example 3 90.1% 1.05%Example 4 91.4% 1.04% Example 5 98.6% 2.05% Example 6 91.3% 5.04%Example 7 90.2% 9.97% Example 8 97.3% 1.98% Example 9 95.8% 3.08%Example 10 98.0% 3.96% Example 11 76.1% 0.30% Example 12 92.9% 1.99%Example 13 90.1% 2.04% Example 14 90.4% 2.04% Comparative 52.4% noExample 1

Table 1 shows that the entrapment efficiency of the microspheres canreach to 90% and above if the goserelin microspheres were prepared fromgoserelin pretreated by Poloxamer/PEG and the content by weight ofPoloxamer/PEG in the goserelin microspheres is above 1%. The entrapmentefficiency of the microspheres containing no poloxamer/PEG is about 50%.

Test Example 2 In Vivo Release Test of Goserelin Microspheres ComprisingPoloxamer/PEG with Different Contents and No Poloxamer/PEG

Test Materials:

Test drugs: goserelin microspheres prepared according to Examples 11, 4,5, 6 and 7, which contain 0.3% (w/w), 1% (w/w), 2%(w/w), 5% (w/w), 10%(w/w) of Poloxamer 188, respectively, goserelin microspheres preparedaccording to Example 12, which contain 2% PEG6000. PLGA used in thesamples are PLGA (50/50, 0.14, 7,200).

Control group: goserelin microspheres containing no poloxamer/PEG andwith a drug loading amount of about 2.42% prepared according toComparative Example 1.

Experimental Animals:

-   -   SD Rats (Shandong Luye Pharmaceutical Co., Ltd. Animal Room).

Test Instruments:

a QTRAP5500 mass spectrometer fitted with an ionspray ionization source(Applied Biosystem, Inc.);

an Agilent 1290 high performance liquid chromatography system comprisinga dual infusion pump, an autosampler and a column oven;

an Anke TGL-16G Feige desk centrifuge, (ShangHai Anting ScientificInstrument Factory); and

a Turbo Vap LV pressure blowing concentrator, (Biotage, Inc).

Test Method:

-   -   a) Experimental animals: male SD rats with body weight of 190±10        g, 4 per group;    -   b) Route of administration and dose: intramuscular injection at        a dose of 0.9 mg/per rat and at a volume of 0.5 mL/per rat.    -   c) Blood sampling time: blood samples were collected before (at        0 h) and after administration at 1 h, 6 h, 1 d, 4 d, 7 d, 9 d,        11 d, 13 d, 15 d, 17 d, 19 d, 23 d and 28 d, respectively.    -   d) Determination of biological samples: the free drug        concentration of goserelin in blood plasma was determined by an        LC-MS/MS method;    -   e) Data processing: DAS 2.0 software.

Results are shown in Table 2 and FIG. 1.

TABLE 2 Blood concentrations of goserelin at different time afterintramuscular injection to rats (ng/mL) Time Comparative Example (Day)Example 1 11 Example 4 Example 5 Example 6 Example 7 Example 12 0 0 0 00 0 0 0 0.04 150.25 31.23 30.60 51.12 43.32 42.57 43.02 0.25 6.17 1.242.10 2.12 2.71 1.43 1.58 1 1.58 1.15 1.81 1.13 2.54 0.95 2.86 4 4.424.02 3.62 5.82 6.24 4.98 5.72 7 4.74 3.50 6.45 9.27 10.87 10.29 10.52 95.60 7.57 8.86 10.96 8.96 10.44 10.48 11 1.37 4.72 5.25 6.78 5.48 5.896.02 13 0.24 3.76 2.91 3.12 2.39 3.46 1.95 15 0.02 2.51 2.12 2.63 1.802.10 1.32 17 0 1.51 1.51 1.65 1.12 1.13 0.73 19 0 0.38 0.38 0.31 0.730.13 0.20 23 0 0.24 0.24 0.20 0.22 0.28 0.19 28 0 0 0 0.02 0.01 0.020.01 AUC 1542.1 1661.4 1875.2 2455.7 2401.9 2308.7 2338.5 (ng/ml * h)

The results show that the goserelin microspheres released drugsimmediately after administration, and the AUC of the goserelinmicrospheres containing poloxamer/PEG was significantly higher thanthose containing no poloxamer/PEG. Thus, the presence of poloxamer/PEGin the goserelin microspheres was shown to increase the in vivobioavailability. Further, the bioavailability of the microspheres wasshown to be relevant to the content of poloxamer/PEG in themicrospheres. When the content of Poloxamer/PEG in goserelinmicrospheres is above 1%, the bioavailability of the goserelinmicrospheres increases more than 20% compared with the goserelinmicrospheres containing no poloxamer/PEG.

Example 15

92 mg of goserelin acetate (containing 80 mg of goserelin as measured)and 6 mg of Poloxamer 188 were weighed and dissolved in 4 ml of water toform a clear solution, and then the solution was freeze-dried so as toobtain a mixture of solid powder. 1.902 g PLGA (50/50, 0.20, 16,000) wasweighed and dissolved in 8 ml of dichloromethane to form an oil phase;and then the pretreated drug mixture of solid powder was added into theoil phase, and subjected to emulsification in a high shearing emulsifier(6,500 rpm, 3 min) so as to obtain a s/o primary emulsion. The primaryemulsion was added into 1,000 ml of a 1.0% PVA solution at 6° C. underhomogenization at 1,800 rpm, and then it was homogeneously emulsifiedfor 2 min to obtain an S/O/W double emulsion. The double emulsion wasstirred to volatilize and remove the organic solvent; the residue waswashed and freeze-dried to obtain powdery microspheres. The microsphereshad a drug loading amount of 2.76% and an entrapment efficiency of74.6%.

Example 16

92 mg of goserelin acetate (containing 80 mg of goserelin as measured)and 20 mg of Poloxamer 188 were weighed and dissolved in 10 ml of waterto form a clear solution, and then the solution was freeze-dried so asto obtain a mixture of solid powder. 1.888 g PLGA (50/50, 0.20, 16,000)was weighed and dissolved in 8 ml of dichloromethane to form an oilphase; and then the pretreated drug mixture of solid powder was addedinto the oil phase, and subjected to emulsification in a high shearingemulsifier (6,500 rpm, 3 min) so as to obtain a s/o primary emulsion.The primary emulsion was added into 1,000 ml of a 1.0% PVA solution at6° C. under homogenization at 1,800 rpm, and then it was homogeneouslyemulsified for 2 min to obtain an S/O/W double emulsion. The doubleemulsion was stirred to volatilize and remove the organic solvent; theresidue was washed and freeze-dried to obtain powdery microspheres. Themicrospheres had a drug loading amount of 3.49% and an entrapmentefficiency of 90.1%.

Example 17

92 mg of goserelin acetate (containing 80 mg of goserelin as measured)and 39 mg of Poloxamer 188 were weighed and dissolved in 10 ml of waterto form a clear solution, and then the solution was freeze-dried so asto obtain a mixture of solid powder. 1.870 g PLGA (50/50, 0.20, 16,000)was weighed and dissolved in 8 ml of dichloromethane to form an oilphase; and then the pretreated drug mixture of solid powder was addedinto the oil phase, and subjected to emulsification in a high shearingemulsifier (6,500 rpm, 3 min) so as to obtain a s/o primary emulsion.The primary emulsion was added into 1,000 ml of a 1.0% PVA solution at6° C. under homogenization at 1,800 rpm, and then it was homogeneouslyemulsified for 2 min to obtain an S/O/W double emulsion. The doubleemulsion was stirred to volatilize and remove the organic solvent; theresidue was washed and freeze-dried to obtain powdery microspheres. Themicrospheres had a drug loading amount of 3.67% and an entrapmentefficiency of 95.7%.

Example 18

92 mg of goserelin acetate (containing 80 mg of goserelin as measured)and 70 mg of Poloxamer 188 were weighed and dissolved in 4 ml of waterto form a clear solution, and then the solution was freeze-dried so asto obtain a mixture of solid powder. 1.838 g PLGA (50/50, 0.20, 16000)was weighed and dissolved in 8 ml of dichloromethane to form an oilphase; and then the pretreated drug mixture of solid powder was addedinto the oil phase, and subjected to emulsification in a high shearingemulsifier (6,500 rpm, 3 min) so as to obtain a s/o primary emulsion.The primary emulsion was added into 1000 ml of a 1.0% PVA solution at 6°C. under homogenization at, rpm, and then it was homogeneouslyemulsified for 2 min to obtain an S/O/W double emulsion. The doubleemulsion was stirred to volatilize and remove the organic solvent; theresidue was washed and freeze-dried to obtain powdery microspheres. Themicrospheres had a drug loading amount of 3.74% and an entrapmentefficiency of 99.3%.

Example 19

92 mg of goserelin acetate (containing 80 mg of goserelin as measured)and 121 mg of Poloxamer 188 were weighed and dissolved in 4 ml of waterto form a clear solution, and then the solution was freeze-dried so asto obtain a mixture of solid powder. 1.786 g PLGA (50/50, 0.20, 16,000)was weighed and dissolved in 8 ml of dichloromethane to form an oilphase; and then the pretreated drug mixture of solid powder was addedinto the oil phase, and subjected to emulsification in a high shearingemulsifier (6,500 rpm, 3 min) so as to obtain a s/o primary emulsion.The primary emulsion was added into 1,000 ml of a 1.0% PVA solution at6° C. under homogenization at 1,800 rpm, and then it was homogeneouslyemulsified for 2 min to obtain an S/O/W double emulsion. The doubleemulsion was stirred to volatilize and remove the organic solvent; theresidue was washed and freeze-dried to obtain powdery microspheres. Themicrospheres had a drug loading amount of 3.38% and an entrapmentefficiency of 91.2%.

Example 20

92 mg of goserelin acetate (containing 80 mg of goserelin as measured)and 201 mg of Poloxamer 188 were weighed and dissolved in 10 ml of waterto form a clear solution, and then the solution was freeze-dried so asto obtain a mixture of solid powder. 1.693 g PLGA (50/50, 0.20, 16,000)was weighed and dissolved in 8 ml of dichloromethane to form an oilphase; and then the pretreated drug mixture of solid powder was addedinto the oil phase, and subjected to emulsification in a high shearingemulsifier (6,500 rpm, 3 min) so as to obtain a s/o primary emulsion.The primary emulsion was added into 1,000 ml of a 1.0% PVA solution at6° C., under homogenization at 1,800 rpm, and then it was homogeneouslyemulsified for 2 min to obtain an S/O/W double emulsion. The doubleemulsion was stirred to volatilize and remove the organic solvent; theresidue was washed and freeze-dried to obtain powdery microspheres. Themicrospheres had a drug loading amount of 3.51% and an entrapmentefficiency of 90.1%.

Example 21

92 mg of goserelin acetate (containing 80 mg of goserelin as measured)and 70 mg of PEG6000 were weighed and dissolved in 4 ml of water to forma clear solution, and then the solution was freeze-dried so as to obtaina mixture of solid powder. 1.838 g PLGA (50/50, 0.20, 16,000) wasweighed and dissolved in 8 ml of dichloromethane to form an oil phase;and then the pretreated drug mixture of solid powder was added into theoil phase, and subjected to emulsification in a high shearing emulsifier(6,500 rpm, 3 min) so as to obtain a s/o primary emulsion. The primaryemulsion was added into 1,000 ml of a 1.0% PVA solution at 6° C. underhomogenization at 1,800 rpm, and then it was homogeneously emulsifiedfor 2 min to obtain an S/O/W double emulsion. The double emulsion wasstirred to volatilize and remove the organic solvent; the residue waswashed and freeze-dried to obtain powdery microspheres. The microsphereshad a drug loading amount of 3.57% and an entrapment efficiency of90.7%.

Comparative Example 2

92 mg of goserelin acetate (containing 80 mg of goserelin as measured)was weighed and dissolved in 4 ml of water to form a clear solution, andthen the solution was freeze-dried so as to obtain a mixture of solidpowder. 1.908 g PLGA (50/50, 0.20, 16,000) was weighed and dissolved in8 ml of dichloromethane to form an oil phase; and then the pretreateddrug mixture of solid powder was added into the oil phase, and subjectedto emulsification in a high shearing emulsifier (6,500 rpm, 3 min) so asto obtain a s/o primary emulsion. The primary emulsion was added into1,000 ml of a 1.0% PVA solution at 6° C. under homogenization at 1,800rpm, and then it was homogeneously emulsified for 2 min to obtain anS/O/W double emulsion. The double emulsion was stirred to volatilize andremove the organic solvent; the residue was washed and freeze-dried toobtain powdery microspheres. The microspheres had a drug loading amountof 2.86% and an entrapment efficiency of 57.1%.

Test Example 3 In Vivo Release Test of Goserelin Microspheres ComprisingPoloxamer/PEG with Different Contents and No Poloxamer/PEG

Test Materials:

Test drugs: goserelin microspheres prepared according to Examples 15,16, 17, 18, 19 and 20, which contain 0.3% (w/w), 1% (w/w), 2% (w/w),3.5% (w/w), 6.0% (w/w) and 10% (w/w) of Poloxamer 188, respectively.Goserelin microspheres prepared according to Example 21, which contain3.5% PEG6000. Polymers used in the samples are all PLGA (50/50, 0.20,16,000).

Control group: goserelin microspheres containing no poloxamer/PEG andwith a drug loading amount of about 2.74% prepared according toComparative Example 2.

Experimental Animals:

SD rats (Shandong Luye Pharmaceutical Co., Ltd Animal Room).

Test Instruments:

a QTRAP5500 mass spectrometer fitted with an ionspray ionization source(Applied Biosystem, Inc.);

an Agilent 1290 high performance liquid chromatography system comprisinga dual infusion pump, an autosampler and a column oven;

an Anke TGL-16G Feige desk centrifuge, (ShangHai Anting ScientificInstrument Factory); and

a Turbo Vap LV pressure blowing concentrator, (Biotage, Inc).

Test Method:

-   -   a) Experimental animals: male SD rats with body weight of 190±10        g, 4 per group:    -   b) Route of administration and doses: intramuscular injection at        a dose of 0.9 mg/per rat and at a volume of 0.5 mL/per rat.    -   c) Blood sampling time: blood samples were collected before (at        0 h) and after administration at 1 h, 6 h, 1 d, 4 d, 7 d, 9 d,        11 d, 13 d, 15 d, 17 d, 19 d, 23 d and 28 d, respectively.    -   d) Determination of biological samples: the free drug        concentration of goserelin in the blood plasma was determined by        an LC-MS/MS method;    -   e) Data processing: DAS 2.0 software.

Results are shown in Table 3 and FIG. 2.

TABLE 3 Blood concentrations of goserelin at different time afterintramuscular injection to rats (ng/mL) Time Example Example ExampleExample Example Example Example (Day) Example 2 15 16 17 18 19 20 21 0 00 0 0 0 0 0 0 0.04 117.21 26.91 27.48 34.31 49.32 43.87 39.31 38.51 0.2512.14 1.69 2.38 2.12 3.41 3.28 2.57 1.59 1 2.14 1.41 2.04 1.13 2.33 1.473.24 1.97 4 4.42 5.21 4.87 5.82 5.21 5.96 5.24 6.07 7 3.87 4.54 5.988.2.7 12.34 9.89 7.21 7.28 9 4.69 5.18 6.41 9.96 10.24 7.54 9.84 10.2411 2.07 4.08 6.54 6.78 5.07 5.28 5.17 4.46 13 0.16 2.94 3.54 3.12 2.173.67 2.47 2.77 15 0.01 2.04 2.01 2.63 1.69 2.46 1.97 2.31 17 0 1.11 1.371.35 0.97 1.08 0.54 0.89 19 0 0.74 0.49 0.62 0.81 0.31 0.36 0.67 23 00.08 0.34 0.19 0.34 0.17 0.21 0.21 28 0 0 0 0.05 0.02 0.01 0.01 0.01 AUC1470.38 1605.06 1916.41 2305.88 2470.51 2260.72 2111.89 2159.99 (ng/ml *h)

The results show that the goserelin microspheres released drugsimmediately after administration, and the AUC of the goserelinmicrospheres containing poloxamer/PEG was significantly higher thanthose containing no poloxamer/PEG. Thus, the presence of poloxamer/PEGin goserelin microspheres was shown to increase the in vivobioavailability. Further, the bioavailability of the microspheres wasshown to be relevant to the content of poloxamer/PEG in themicrospheres. When the content of Poloxamer/PEG in goserelinmicrospheres is above 1%, the bioavailability of the goserelinmicrospheres increases more than 20% compared with the goserelinmicrospheres containing no poloxamer/PEG.

Example 22

9.2 g of goserelin acetate (containing 8 g of goserelin as measured) and7.0 g of Poloxamer 188 were weighed and dissolved in 400 ml of water toform a clear solution, and then the solution was freeze-dried so as toobtain a mixture of solid powder (measuring the content of acetic acidin the intermediate, i.e., the freeze-dried powder of goserelin acetateand poloxamer; the test method is shown in test Example 4). 183.8 g PLGA(50/50, 0.20, 16,000) was weighed and dissolved in 800 ml ofdichloromethane to form an oil phase; and then the pretreated drugmixture of solid powder was added into the oil phase, and subjected toemulsification in a high shearing emulsifier (6,500 rpm, 3 min) so as toobtain a s/o primary emulsion. The primary emulsion was added into 100 Lof a 1.0% PVA solution at 6° C. under homogenization at 1,800 rpm, andthen it was homogeneously emulsified for 2 min to obtain an S/O/W doubleemulsion. The double emulsion was stirred to volatilize and remove theorganic solvent; the residue was washed and freeze-dried to obtainpowdery microspheres. The microspheres had a drug loading amount of3.73% and an entrapment efficiency of 97.4%.

Example 23

9.2 g of goserelin acetate (containing 8 g of goserelin as measured) and7.0 g of Poloxamer 188 were weighed and dissolved in 400 ml of water toform a clear solution, and then the solution was freeze-dried so as toobtain a mixture of solid powder (measuring the content of acetic acidin the intermediate, i.e., the freeze-dried powder of goserelin acetateand poloxamer; the test method is shown in test Example 4; extending thefreeze-drying time and raising temperature until the detected content ofacetic acid is no more than 0.5%). 183.8 g PLGA (50/50, 0.20, 16,000)was weighed and dissolved in 800 ml of dichloromethane to form an oilphase; and then the pretreated drug mixture of solid powder was addedinto the oil phase, and subjected to emulsification in a high shearingemulsifier (6,500 rpm, 3 min) so as to obtain a s/o primary emulsion.The primary emulsion was added into 100 L of a 1.0% PVA solution at 6°C. under homogenization at 1,800 rpm, and then it was homogeneouslyemulsified for 2 min to obtain an S/O/W double emulsion. The doubleemulsion was stirred to volatilize and remove the organic solvent; theresidue was washed and freeze-dried to obtain powdery microspheres. Themicrospheres had a drug loading amount of 3.71% and an entrapmentefficiency of 99.6%.

Example 24

9.2 g of goserelin acetate (containing 8 g of goserelin as measured) and7.0 mg of Poloxamer 188 were weighed and dissolved in 400 ml of 0.1% ofammonia water to form a clear solution, and then the solution wasfreeze-dried so as to obtain a mixture of solid powder (measuring thecontent of acetic acid in the intermediate, i.e., the freeze-driedpowder of goserelin acetate and poloxamer; the test method is shown intest Example 4). 183.8 g PLGA (50/50, 0.20, 16,000) was weighed anddissolved in 800 ml of dichloromethane to form an oil phase; and thenthe pretreated drug mixture of solid powder was added into the oilphase, and subjected to emulsification in a high shearing emulsifier(6,500 rpm, 3 min) so as to obtain a s/o primary emulsion. The primaryemulsion was added into 100 L of a 1.0% PVA solution at 6° C. through aninjector under homogenization at 1,800 rpm, and then it washomogeneously emulsified for 2 min to obtain an S/O/W double emulsion.The double emulsion was stirred to volatilize and remove the organicsolvent; the residue was washed and freeze-dried to obtain powderymicrospheres. The microspheres had a drug loading amount of 3.73% and anentrapment efficiency of 99.4%.

Test Example 4 Influence of Different Content of Acetic Acid inGoserelin Microspheres on the In Vivo Release Amount

Test method: determine with gas chromatography [Appendix VE, Method 3,of Chinese Pharmacopoeia (second part, 2000 Edition)].

Chromatographic Conditions and System Suitability Test

The column was a 10 meters long capillary column with inner diameter of0.32 mm, and the inner layer was coated with 0.33 μm of FFAP-CB fusedsilica. The chromatographic conditions were set as follows:

Injection temperature: 220° C.;

Detector temperature: 250° C.;

Split ratio: 100:1;

The column temperature program was set as follows:

-   -   (1) starting at temperature of 50° C., and remained the same for        0.10 minutes;    -   (2) increasing the temperature at a rate of 30° C./min;    -   (3) the final temperature of 230° C., and remained for 5        minutes;

Injection volume: 1 μl;

The number of theoretical plates: calculated according to acetate peakand should not be less than 5000. The resolution of acetic acid peak andthe internal standard peak should conform to the specifications.

Determination of the Correction Factor

1.0 ml n-hexadecane was precisely weighted and dissolved in 30 ml ofdimethylformamide in a 50 ml volumetric flask, diluted to volume andshaken well to be used as internal standard solution. 625 mg of aceticacid reference was precisely weighted and dissolved in dimethylformamidein a 100 ml volumetric flask, diluted to volume and shaken well beforeuse. 10 ml of the aforesaid solution was transferred into a 100 mlvolumetric flask. 5 ml of the internal standard solution was added. Thesolution was dissolved with dimethylformamide and diluted to volume andshaken well. 1 μl of the solution was injected into gas chromatographyand injected continually for 3-5 times. The correction factor wascalculated according to average peak area.

Test Sample Preparation and Measurement

About 50 mg of goserelin microspheres prepared according to Example 22,23 and 24 was weighted and transferred into a 2 ml volumetric flask,into which 1 ml of dimethylformamide was added to dissolve the sample.100 μl internal standard solution was added precisely and then the flaskwas brought to final volume with dimethylformamide and shaken well. 1 μlof the sample was injected into gas chromatograph. The result wascalculated by internal standard method.

Results are Shown in Table 4.

TABLE 4 Test substance Example 22 Example 23 Example 24 The content ofacetic 2.79% 0.47% 0.36% acid in the solid powder The content of acetic0.39% 0.0057% 0.0048% acid in the microspheres

The goserelin microspheres in Example 22, 23 and 24 were studied forstability by conducting in vivo release test between samples immediatelyafter prepared and samples stored for 6 months at temperature of 25° C.and humidity of 75%. The test method is showed in Test Example 2 andresults are shown in Table 5 and FIGS. 3 and 4.

TABLE 5 Blood concentrations of goserelin at different time afterintramuscular injection to rats (ng/mL) Example 22 Example 23 Example 24Example 22 (25° C./75%, 6 Example 23 (25° C./75%, 6 Example 24 (25°C./75%, 6 Time (Day) (0 month) months) (0 month) months) (0 month)months) 0 0 0 0 0 0 0 0.04 51.41 33.07 47.38 48.37 49.25 50.07 0.25 4.215.37 5.37 5.39 5.37 4.67 1 3.17 3.97 4.37 5.27 4.06 3.29 4 4.07 3.033.41 3.29 3.97 5.81 7 11.58 9.79 10.97 9.58 11.37 10.67 9 9.47 6.38 9.548.47 8.26 8.67 11 7.59 4.41 7.54 6.98 6.59 4.69 13 4.04 2.76 4.2 5.674.26 5.22 15 1.24 1.86 1.57 2.07 1.89 1.67 17 0.48 1.01 1.06 1.27 1.291.65 19 0.67 0.74 0.59 0.94 0.97 0.57 23 0.17 0.02 0.08 0.13 0.06 0.1628 0.01 0.02 0.01 0.01 0.01 0.01 AUC (ng/ml * h) 2495.7 1989.98 2503.672512.33 2507.09 2520.61

The results show that control the content of acetic acid during theprocess of goserelin acetate microspheres of does not affect the drugloading amount and the entrapment efficiency. However, the in vivo drugrelease amount changed after the stability study (after stored for 6months in condition of temperature of 25° C. and humidity of 75%). Thein vivo drug release amount does not change when the content of aceticacid in goserelin microspheres is less than 0.01%, while the in vivodrug release amount of the goserelin microspheres decreases by more than20% when the content of acetic acid in goserelin microspheres is notcontrolled

Test Example 5 Measurement of the Particle Sizes of Solid Powder in OilPhase and the Particle Sizes of Microspheres Prepared

Test drugs: goserelin microspheres prepared according to Examples 15,16, 17, 18, 19 and 20, which contain 0.3% (w/w), 1% (w/w), 2% (w/w),3.5% (w/w), 6.0%(w/w) and 10% (w/w) of Poloxamer 188, respectively;goserelin microspheres prepared according to Example 21, which contain2% (w/w) of PEG6000. PLGA used in the samples are 50/50, 0.20, 16,000.

Control group: goserelin microspheres containing no poloxamer/PEG andwith a drug loading amount of about 2.74% prepared according toComparative Example 2.

Test method: According to the related regulations on particle size andparticle size distribution measurement method [The third method inAppendix XIX E of Chinese Pharmacopoeia (second part, 2010 Edition)],0.1% Tween 20 solution was used as dispersing agent. About 120 ml Tween20 solution was transferred into the sample dispersion device of aparticle size analyzer, and the rotational speed controller was adjustedso that the stirring was at 2,100 rpm. The background of dispersingagent was measured first. Then 0.1 ml of primary emulsion and 50 mg offreeze-dried microspheres powder were added into the dispersing agent.After the samples were distributed evenly, the particle sizes weremeasured in parallel for three times and the average numbers were taken.

Results are shown in Table 6.

TABLE 6 Primary emulsion Microspheres Microspheres Example No. d (0.5)D(4, 3) (span) Example 15 3.44 μm 157.4 2.67 Example 16 0.374 μm 117.80.89 Example 17 0.191 μm 94.7 0.74 Example 18 0.067 μm 88.5 0.33 Example19 0.257 μm 99.7 0.97 Example 20 0.547 μm 126.9 0.84 Comparative 6.79 μm196.3 3.56 Example 2

The results show that when preparing the microspheres, control ofparticle sizes of primary emulsion had significant effect on theparticle sizes of the microspheres. When the particle sizes of theprimary emulsion were controlled, the span of the goserelin microspherescould be ten times less than the situation when the particle sizes ofthe primary emulsion were not controlled. Therefore by controlling theparticle sizes of the primary emulsion, more uniform sizes ofmicrospheres product could be obtained.

What is claimed is:
 1. A pharmaceutical composition of sustained release goserelin microspheres, comprising (a) goserelin or a salt thereof, (b) poly(lactide-co-glycolide), and (c) poloxamer or polyethylene glycol (PEG).
 2. The pharmaceutical composition according to claim 1, wherein the poloxamer is poloxamer 188 or poloxamer
 407. 3. The pharmaceutical composition according to claim 1, wherein the PEG is PEG 2000, PEG 4000 or PEG
 6000. 4. The pharmaceutical composition according to claim 2, wherein the content by weight of poloxamer or PEG is within a range from 1% to 10%.
 5. The pharmaceutical composition according to claim 1, wherein the content by weight of the goserelin or a salt thereof is within a range from 1% to 10%.
 6. The pharmaceutical composition according to claim 1, wherein the molar ratio of lactide to glycolide in the poly(lactide-co-glycolide) is within a range from 90:10 to 10:90.
 7. The pharmaceutical composition according to claim 1, wherein the poly(lactide-co-glycolide) has an intrinsic viscosity of 0.10-0.40 dl/g.
 8. The pharmaceutical composition according to claim 1, wherein the weight average molecular weight of the poly(lactide-co-glycolide) is 4,000-45,000 Dalton.
 9. The pharmaceutical composition according to claim 1, wherein the weight content of the poly(lactide-co-glycolide) is 80-98%.
 10. The pharmaceutical composition according to claim 1, further comprising (d) acetic acid.
 11. The pharmaceutical composition according to claim 10, wherein the weight content of the acetic acid is less than 0.1%.
 12. The pharmaceutical composition according to claim 11, wherein the weight content of the acetic acid is less than 0.01%.
 13. The pharmaceutical composition according to claim 1, wherein the weight content of the goserelin is 1-10%; wherein the weight content of the poly(lactide-co-glycolide) is 80-98%; and wherein the weight content of the poloxamer or PEG is 1-10%.
 14. The pharmaceutical composition according to claim 1, wherein the weight content of the goserelin is 1-8%; wherein the weight content of the poly(lactide-co-glycolide) is 86-98%; and wherein the weight content of the poloxamer or PEG is 1-6%.
 15. The pharmaceutical composition according to claim 1, wherein the weight content of the goserelin is 1-5%; wherein the weight content of the poly(lactide-co-glycolide) is 91-98%; and wherein the weight content of poloxamer or PEG is 1-4%.
 16. A method for preparing the pharmaceutical composition of claim 1, wherein the composition is prepared by a solid-in-oil-in-water (S/O/W) emulsion-solvent evaporation method, comprising pre-treating goserelin acetate with poloxamer or PEG, and then adding the pretreated goserelin acetate to an oil phase comprising poly(lactide-co-glycolide).
 17. The method of claim 16, wherein the poloxamer or PEG pre-treated goserelin acetate in the oil phase has a particle size d (0.5) of 0.01-2 μm.
 18. A method for treating prostate cancer, sexual precocity, adenomyosis, female infertility, or hysteromyoma in a subject in need thereof, comprising administering to the subject an effective amount of the pharmaceutical composition of claim
 1. 